%0 Journal Article %A Britz, M. L. %A Wilkinson, R. G. %T Partial Purification and Characterization of Two Enzymes Involved in Isovaleric Acid Synthesis in Clostridium bifermentans %D 1983 %J Microbiology, %V 129 %N 10 %P 3227-3237 %@ 1465-2080 %R https://doi.org/10.1099/00221287-129-10-3227 %I Microbiology Society, %X Conversion of leucine to isovaleric acid by Clostridium bifermentans is achieved by the action of at least two enzymes. One is a transaminase producing α-ketoisocaproic acid, which was purified 30-fold from osmotic lysates of late-exponential phase cells by repeated chromatography on DEAE-Sepharose C16B and Sephacryl S300: this represented a 147-fold purification of activity found in sonically disrupted cells. This enzyme had an apparent molecular weight of approximately 190000 and was composed of six identically sized sub-units (molecular weight 31000 ± 1000). Transamination required pyridoxal phosphate and pyruvate and was optimal at pH 8·6: the apparent K m for leucine was 7·0 mm. Activity was totally inhibited by 1 mm-p-chloromercuribenzoate and partially inhibited by other thiol reagents. The second enzyme decarboxylated α-ketoisocaproic acid to form isovaleric acid and was also partially purified by chromatography en DEAE-Sepharose C16B and Sephacryl S300. It has an apparent molecular weight of 240000 and required FAD and coenzyme A for activity; the K m for α-ketoisocaproic acid was 4·2 mM and activity was optimal around pH 8·0. This enzyme was a flavoprotein with absorption maxima at 280, 320 and 400 nm, and a fluorescent maximum at 500 nm. The prosthetic group. FAD, dissociated from the protein during purification resulting in an inactive apoenzyme which was only partially re-activated by FAD. Activity was completely inhibited by several thiol reagents tested at 1 mm. %U https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-129-10-3227